Poly-5-halogeno-2{40 -halogeno-2{40 -deoxyuridylic acids

ABSTRACT

Poly-5-halogeno-2&#39;&#39;-halogeno-2&#39;&#39;-deoxyuridylic acids in which the 5-halogeno substituent is fluoro, chloro, bromo, or iodo and the 2&#39;&#39;-halogeno group is fluoro, chloro, or bromo are useful as inhibitors of avian myeloblastosis virus RNA-dependent DNA polymerase.

United States Patent [191 Kotick et al.

POLY-S-HALOGENO-Z '-HALOGENO-2 DEOXYURIDYLIC ACIDS Inventors: Michael Paul Kotick, Elkhart;

Chimanbhai P. Patel, Mishawaka; Joseph L. Colbourn, Elkhart; Robert J. Erickson, Elkhart, all of lnd Assignee: Miles Laboratories, Inc., Elkhart,

Ind.

Filed: Dec. 6, 1973 App]. No.: 422,403

US. Cl 260/2115 R; 424/180 Int. Cl."'- C07 19/10 Field of Search 260/2115 R [451 Sept. 16, 1975 56] References Cited UNITED STATES PATENTS 3,687,93l 8/1972 Vcrhcydcn at al v. 260/21 [.5 R 3,792,039 2/1974 Erickson 6! a], 260/21 1.5 R

Primary ExaminerJohnnie R. Brown Alromey, Agent, or Firm-Myron B. Sokolowski [57] ABSTRACT 13 Claims, No Drawings 3,965 953 1 2 POLY-S-HALOGENO-Z'-HALOGENO-2'- SUMMARY OF THE INVENTION DEOXYURIDYUC ACIDS The subject matter of this invention involves certain BACKGROUND OF THE INVENTION cligoand poly-S-halogeno-Z'-halogeno-2'- deoxyuridylic acids which are polymerized by enzymatic catalysis from corresponding 5-halogeno-2'- The followi"! Poll/nucleotides have been leponed in halogeno-2'-deoxyuridine diphosphates. In the mono- Description of the Prior Art the literature: mer and polymer, the S-halogeno substituent is fluoro.

P yy y acid L at chloro, bromo. or iodo, and the 2'-halogeno group is ochem. Biophys. Res. Commun., 46: 1 153 flu ro. hl or bmmo.

(1972)]; 10 Specifically. this invention pertains to compounds poly-2-fluoro-2-deoxycytidylic acid ll-lendler. et h i h f ula (I) Htj HN Y HO... o

Ci -Cl-l H-N Y l-lO P=0 l I) O CI-l 0 O X .1 n

H- Y HO-P =0 N I al., Abstracts, l62nd National Meeting, American 50 in which: Y is fluoro, chloro, bromo, or iodo; X is Chemical Society, Sept. l97l Abstract No. Biol. fluoro, chloro, or bromo; and n is an integer having a 20]; value of from zero to infinity. In the case where n has poly-2'-chloro-2'-deoxyuridylic and cytidylic acids the value of zer 1 ep esents di-5-hal0gen0-2'- [H0bbs, et a]., Biochemistry, l l: 4336 (l972)]; halogeno-2'-deoxy-S-uridylic acid. p0ly-2-azido-2'-deoxyuridylic acid [Torrence, et al., 55 Specific examples of compounds represented by (1) Biochemistry, 12: 3962 (1973)]; and include the following:

poly-2'-amino-2'-deoxyuridylic acid [Hobbs, et al., Biochem. Biophys. Res. Commun., 46: l509 (1972)].

Several 2'-O-methylated or 2'-O-ethylated pyrimidine and purine polynucleotides have been reported as have certain 2'-0-acety] polynucleotides. To date, the only altered synthetic polynucleotide which has been demonstrated to be an inhibitor of reverse transcriptase, poIyS-bromo-2-chloro-2'-deoxyuridylic acid. aside from the work reported by Erickson et al., has poly-5-bromo-2-bromo-2-deoxyuridylic acid. been by poly-2'-0-methyl adenylic acid [Tennant, Ken- 10. poly-5-iodo-2'-fluoro-2'-deoxyuridylic acid. ney. Tuominen, Nature New Biology, 238: 51 1972)]. l l. poly-5-iodo-2-chloro-2'-deoxyuridylic acid.

. poly-S-fluoro-Z-chloro-2-deoxyuridylic acid.

. poly-5-fluoro-2'-bromo-2-deoxyuridylic acid. poly-5-chloro-2'-fluoro-2'-deoxyuridylic acid. poly-S-chloro-Z'-chloro-2'-deoxyuridylic acid. poly-5-chloro-2'-bromo-2'-deoxyuridylic acid. poly-5-bromo-2'-fluoro-2'-deoxyuridylic acid.

l2. poly-iodo-2'-bromo-2'-deoxyuridylic acid.

The oligoand poly-nucleotides represented by (l) are prepared by enzymatic catalysis from corresponding uridine-5'-diphosphates having the formula H -N I Y i 1 no J o- I o 411.. 0 (ll H HO HO X

uo-cn O [I I) in which the X and Y substituents have the same mean ing as defined in Formula (I). Compounds (III) in turn are prepared according to the following reaction sequence.

HOCHZ 0 (III) HO x In the above series of reactions, X and Y are groups as defined above for Formula (I The synthesis of 2.2 -anhydrol [3-D- arabinofuranosyl) uracil (Formula IV) and its conversion to 2'-halogeno2'-deoxyuridine substituted at either the 5' or 4' position (Formula V) can be performed according to published methods (Codington et al., J. Org. Chem.. 29: 558 [19641;Codington et al. J. Org. Chem.. 29: 564 [I974]; Doerr et al., J. Org. Chem.. 32: 1462 [I967]; and Cushley et al., Can. J. Chem.. 46: H3] [1968]). The 2'-fluoro-2'- deoxycytidine analogues of Formula (II), however, were prepared by the procedure of Hendler and coworkers (Abstracts l62nd National Meeting of the American Chemical Society. Sept. 197 I. No. Biol. 20) or of Shannahoff and Sanchez (J. Org. Chem., 38: 593 1973 The conversion of Formula (V) to Formula (Ill) can be accomplished by the following methods: the 5 fluoro analogues were prepared according to the reaction described by Robins and Naik (J. Amer. Chem. Soc. 93: 5277 [1971 Chem. Comm. I8: [1972]); 5- chloro and 5-bromo derivatives were made by the pathway described by Fukuhyana and Visser (J. Biol. Chem., l9(): [l95l 1); and S-iodo compounds were synthesized according to Prusoff et al.. (Cancer Res, 13: 22] [1953]; Biochem. Biophys. Acta. 32: 295 1959] Compounds represented by Formula (I) are useful as inhibitors of the RNA-dependent DNA polyermase of oncogenic RNA tumor viruses. For example, poly5- bromo-2'-chloro-2-deoxyuridylic acid and poly-5- chloro-2-chloro-2-deoxyuridylic acids are approximately twice as potent with regard to inhibition of the RNA-dependent DNA polymerase derived from avian myeloblastosis virus (AMV) than poly-2'-fluoro-2'- dcoxyuridylic acid (Janik et al., Biochem. Biophys. Res. Comm., 46: H53 [1972]).

Compounds represented by Formula (II) are useful in the preparation of the oligoand poly-5 -halogeno-2'- halogeno-2'-deoxyuridylic acids, (l).

DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLES ll 2 5-Halogeno-2-Halogeno-2"Deoxyuridine-5 Diphosphates A mixture of between 2.4 and 3.2 ml of trimethyl phosphate and between 0.65 to 1.2 ml (7 mmoles to 13 mmoles) of distilled phosphorous oxychloride was stirred under anhydrous conditions in an ice-bath for minutes. To the cooled, stirred mixture was added the appropriate nucleoside (l mmole) in a single portion. The nucleoside dissolved after stirring for a short time, and stirring under anhydrous conditions was continued at ice-bath temperature for 6 hours. The reaction mixture was then poured over g of crushed ice. and the resulting solution was stirred with cooling in an ice-bath for minutes. To the cooled, stirred mixture was then added, in equimolar proportion to the phosphorous oxychloride used above, magnesium acetate tetrahydrate l .50 g. to 2.79 g, 7 mmoles to 13 mmoles). The cold reaction mixture was then made basic by the addition of concentrated ammonium hydroxide until the pH was stabilized at about pH 8 (moist pH paper). Stirring and cooling of the mixture was continued for 30 minutes in the ice-bath after which the insoluble Mg( NH4)-PO4XH2O was removed by filtration and washed with water adjusted to pH 8 by the addition of ammonium hydroxide. The combined filtrate and washings were passed through a column of an acidic ion-exchange resin (Dowex 50 H X 4 or X 8, approximately 100 ml volume) and the ultraviolet absorbing, acidic portion of the eluant collected. The column was washed with water to remove all the ultraviolet absorbing material. The combined eluants were then evaporated in vacuo at about to to a thick syrup. The syrup was azeotroped several times with water and finally was dissolved in methanol (20 ml) and tri-n-butylamine (0.25 ml, 1 mmole) added. The methanolic solution was evaporated to dryness, dissolved in dimethylformamide (DMF) (20 ml) and the solution concentrated in high vacuum at 45 to 50 to a small volume. The residue was azeotroped with three 30 ml portions ofdry DMF (distilled in vacuo and kept over 4A Molecular Sieve). The residue was finally dissolved in DMF (20 ml) and to the clear, stirred solution was added l,l -carbonyldiimidazole (324 mg, 2 mmole). The mixture was stirred at room temperature under anhydrous conditions. After 30 minutes. an anhydrous solution of mono-tri-n-butyl ammonium phosphate (4 mmole) in DMF l0 to 15 ml) was added and the reaction mixture stirred under anhydrous conditions overnight at room temperature [6 to 20 hours). The reaction solution was decanted from some insoluble gummy material or clarified by filtration, the gum or collected insoluble material in each case being extracted or washed with additional quantities of DMF. The combined DMF solution containing the product was evaporated under high vacuum at 45 to 50 to a small volume and the residual material taken up in water (50 to I00 ml). The aqueous solution containing the desired nucleotide-5 '-diphosphate was fractionated on an ion-exchange column (DEAE-Sephadex-A-25. bicarbonate form, ml volume) using a linear gradient consisting of 4 L of water in the mixing chamber and 4 L of 0.5 M triethylammonium bicarbonate (pH 7.4-7.8) in the buffer chamber. The major, well resolved, optical density peak containing the diphosphate was concentrated to a small volume and then freeze dried to give the tri-triethylammonium salt of the nucleotide as a foam. The nucleoside-5'-diphosphate so obtained migrated as a single homogeneous spot on thin layer chromatography using cellulose plates with a fluorescence indicator and a solvent system consisting of isobutyric acid-water-concentrated ammonium hydroxide 0.05 M EDTA (l00-56-4-3.2 v/v) or on a cellulose MN 300 polyethyleneimine impregnated with fluorescence indicator (Brinkman Instruments Polygram Cek 300 PEl/UV254) using various concentrations of aqueous LiCl as the developing solvent (02 0.8 M). The following 5-halogeno-2'-halogeno-2'- deoxyuridine-S-diphosphates were prepared in the above manner:

Tri-triethylammonium Molecular Weight O.D. Units of Salt of nucleoside of Triethyl nucleotide 5'-diphosphate -ummonium Salt obtained l) 5-Fluoro-2'-fluoro 727 I788 -2'-deoxyuridine 2] fi-ChlonvT-chloro 760 2925 -2'-dcoxyuridine 3) 5-Bromo-2'-chloro K04 2634 -2'-deoxyuridine 4) 5-lodo-2'chloro 245i I750 -2'-deoxyuridine EXAMPLES l324 Poly-5-Halogeno-2 '-Halogeno-2'-Deoxyuridylic Acids l3. P0ly-5-Bromo-2-Chloro-2'-Deoxyuridylic Acid Polymerization of the substrate nucleoside-5'- diphosphate was accomplished with polynucleotide phosphorylase which was prepared from lzlvr-livricliiu roll. The reaction vessel contained in a total volume ol' li) ml, 0.l M Tris buffer pH 9.0. 4 mM MnCl 97 .umoles of the substrate and the enzyme at a concentra tion of 100 units/ml. The mixture was incubated at 37 and the extent of reaction followed by measurement of the release of inorganic phosphate. After 48 hours at 37. the reaction was terminated by the addition of 0.4 ml of 0.2 M EDTA to the reaction vessel. The reaction mixture was then applied to two chromatographic columns in series. 2.5 cm X 90 cm each containing Sephadex 6-50, and the columns were developed with a solvent consisting of 0.2 M NaCl and 1 mM EDTA. The polymeric material eluted with the void volume. The pooled eluant (40 ml) containing the polymeric material was dialyzed against 0.2 M NaCl and.] mM EDTA. This fraction was then deproteinized in the following manner:

Sufficient l M Tris buffer pH 9.0 was added to make 7 the solution 0.0l M with regard to the buffer. This solution was mixed with an equal volume of phenol previously saturated with a mixture of 0.0! M pH 9.0 Tris buffer and 0.l M NaCl. The mixture was shaken for to 10 mins. and the emulsion which formed broken by low speed centrifugation. if necessary. The phenol layer was again extracted with an equal volume of 0.01 M Tris buffer pH 9.0-0.l M NaCl. The combined aque ous layers after deproteinization were exhaustively dia lyzed against water and the solution remaining in the dialysis tubing freeze dried. The residual material, 36 optical density units (A 277). was relyophilized. Poly 5-bromo-2-chloro-2'-deoxyuridylic acid obtained in this manner had the following characteristics: S l2.7

in a solution containing l M NaCl-phosphate buffer,

pH 7.l of ionic strength 005; A 276.5 nm at pH 7 to pH 4; E nm 7.700 in a solution containing 0.l M NaCl phosphate buffer, pH 7.0 of ionic strength 0.05, based on phosphorus analysis of an aqueous solu tion of the polymer and an E nm 5. l00 which is pH independent and also based on phosphorus analysis of the polymer in water; pK,, 8.38 in a phosphate buffer solution of 0. l5 ionic strength at 25. l4. Poly-S-Chloro-Z'-Chloro-2'-Deoxyuridylic Acid Polymerization was accomplished in a similar mair ner to that described above. Thus, the reaction vessel contained in a total volume of 10 ml. I38 pmoles of the substrate. 4 mM MnCL, 0.l M Tris buffer pH 9.0. 0.] M KCl 0.4 mM EDTA and the enzyme at a concentration of I00 units/ml. The mixture was incubated at 37 and the extent of reaction followed by measurement of the inorganic phosphate released. After 48 hours at 37. the reaction was terminated by the addition of 0.4 ml of 0.2 M EDTA. The reaction mixture was then applied to two chromatographic columns in series. 2.5 cm X 90 cm. each containing Sephadex (L50 and the col umns were eluted with a solvent of 0.2 M NaCl-l mM EDTA. The polymeric material eluted with the void volume. The pooled eluant (52 ml) containing the polymcr was dialyzed against 0.2 M NaCLl mM EDTA. The solution remaining in the dialysis tubing was then deproteinized as described above and the aqueous layer and aqueous wash were combined and again dialyzed against water to give 68 optical density units of polymer. The aqueous solution was lyophilized to give poly-5-chloro2'-chloro-2'-deoxyuridylic acid fluffy. hygroscopic powder. Poly-5-chloro 2'-chloro 2'-dcoxyuridylic acid obtained in this manner had the following characteristics: 5 10.2 in a solution containing l M NaCl-phosphate buffer. pH 7.i. of ionic strength 0.05: A 273.0 nm at pH 7 to pH 4: El 6.500 and E 4.950 in a solution containing (Ll M NaCl-phosphate -phosphate buffer. pH 7.0 of ionic strength 0.05. based on phosphorus analysis of an aqueous solution and an E 4.000 which is pH independent and also based on phosphorus analysis of the polymer in water; pK,, 8.29 in a phosphate buffer solution of 0.15 ionic strength at 25.

The following Examples of 5-ha1ogeno-2'-halogeno- 2'-deoxyuridylic acids are prepared in a similar manner:

poly-5-fluoro2'-fluoro-2'-deoxyuridylic acid. poly-5-fluoro-2'-chloro-2'-deoxyuridylie acid. poly-5-fluor0-2'-bromo2'-deoxyuridylic acid. 18. poly-5-chloro-2'-fluoro-2'-deoxyuridylic acid. 19. poly-5-chloro-2'-bromo-2-deoxyuridylic acid. poly-5-bromo-2'-fluoro-2'-deoxyuridylic acid. poly-5-bromo2'-bromo-2-deoxyuridylic acid. poly-5iodo-2'-fluoro-2'-deoxyuridylic acid.

. poly-5-iodo-2'-chloro-2'-(leoxyuridylic acid.

. poly 5-iodo-2-bromo-2-dcoxyuridylic acid.

iumiuioio be m- O EXAMPLE 25 Inhibition of the RNA-dependent DNA Polymerase from Avian Myeloblastosis Virus (AMV) virus and enzyme purification.

Virus particles were concentrated by ccntrifugation at 60,000 X for mins. onto l002i glycerol cushions. The virus was collected, dialyzcd against buffer (0. l5 M NaCl, 3 mM EDTA. 0.01 M lris-HCI, pH 8.5). layered on a 25-60?! sucrose gradient and centrifuged at 40.00 X g at 4 C for 90 mins. The visible band of virus was removed. dialyzed against 0.l5 M NaCl in 0.01 M lris-HCl. pH 8.5, and stored at C in 1071 glycerol.

The AMV DNA polymerase was isolated from purified virus in the following manner: The virus was dis rupted by exposure to l.()"/( sodium deoxycholate and l.0fi Nonidet P-40 (Shell Chemical Co). centrifuged at 16.000 I g. and the supernatant applied to a Whatman DEAE-ccllulosc DESI column. The active fractions were pooled. diluted. and applied to a Reeve Angel phosphocellulose P-l 1 column. The enzyme samples were stored in 2 mM dithiothreitol and 507? glycerol at 20(.

Assay for RNA-directed DNA polymerase activity.

The polymerization of either dGMP or dTMP using the complementary primer-templates. dipGh 'polytC) or d( p'l) polyi A) respectively. was carried out at 37 C in the following standard reaction mixture. [0 mM TrisHCl buffer (pH 8.5); 7 niM KCl; 2 mM dithiothreitol; l0 mM MgCl 0.6 mM dCTP or dATP; and 0.1 5 mM dCiTP or d'ITP. Tritiated dGTP or dTTP was added at a level of0.0l uCi/ul assay mixture or approve imatcly 20 cpm/pmole. Only the two complementary deoxynuclcoside triphosphatcs were added to each re action. The primcr templates were prepared in a ratio of lzo (wfwl unless stated otherwise.

When the polymerase activity in purified virions was assayed. the particles were first disrupted in 0.29? Nonidct P40 at 1 C for 20 mins. and then added to the assay mixture at a level of 10 pg Pl'OtClll/PLT.

In such experiments, L 1.1.] of enzyme solution polymerized 3.0 pmoles dGMP/min. Trichloroacetic acid insoluble product was collected and washed on nitrocellulose filters. The filters were dried, placed in a to]- uene-based scintillation fluid and assayed in a liquid scintillation spectrometer. Inhibition constants for the poly-S-halogeno-Z '-halogeno-2 'deoxyuridylic acids were obtained from plots of [I] vs l/V,, where [I] is the concentration of the inhibitor and V, is the initial velocity of the uninhibited reaction.

The inhibition constant, K,, for poly-2'-fluoro-2'- deoxyuridylic acid, the reference compounds, was determined to be 40 ng/ml.

The inhibitory constant, K, of poly-5-bromo-2'- chloro-2'-deoxyuridylic acid and poly-5-chloro-2'- chloro-2'-deoxyuridylic acid were determined from a plot of [I] vs l/V, and were found to be approximately ng/ml. Hence, poly-S-bromoand poly-S-chloro-2'- chloro-2-deoxyuridylic acids are equally potent with regard to their inhibitory powers against AMV RNA- directed DNA polymerase and are approximately twice more potent in inhibition than poly-2-fluoro-2'- deoxyuridylic acid.

What is claimed is:

l. A compound having the formula Y HO H- I H0 l :0 A};

Cl-i

Y no.1. ,0 *24 O H-N I Y HO X wherein:

X is selected from the group consisting of fluoro.

chloro, and bromo; Y is selected from the group consisting of fluoro,

chloro, bromo, and iodo; and

n is an integer having a value of from zero to infinity.

2. A compound as in claim I, poly-5-fluoro-2'-fluoro- 2'-deoxyuridylic acid.

3. A compound as in claim 1, poly-5-fluoro-2'- chloro-2-deoxyuridylic acid.

4. A compound as in claim 1, poly-5-fluoro-2'- bromo-Z '-deoxyuridylic acid.

5. A compound as in claim 1, fluoro-2-deoxyuridylic acid.

6. A compound as in claim I, chloro-2'-deoxyuridylic acid.

7. A compound as in claim bromo-Z'deoxyuridylic acid.

8. A compound as in claim fluoro-2'-deoxyuridylic acid.

9. A compound as in claim chloro-2'deoxyuridylic acid.

10. A compound as in claim I, bromo-2'-deoxyuridylic acid.

11. A compound as in claim 1, poly-5-iodo-2'-fluoro- 2'-deoxyuridylic acid.

12. A compound as in claim I, poly-5-iodo-2'-chloropoly-5-chloro-2- poly-5-chloro-2- l, poly-5-chloro-2- l, poly-5-bromo-2'- l, poly-5-bromo-2- poly-S-bromo-Z 2-deoxyuridylic acid.

13. A compound as in claim 1, poly-5-iodo-2'- bromo-2'-deoxyuridylic acid.

UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION PATENT NO. 3,905,953

DATED September 16, 1975 INVENTOR(S) 1 Michael P. Kotick; Chimanbhai P. Patel; Joseph L.

H Colbourn & Robert J. Erickson It rs certrfred that error appears In the above-rdentrfred patent and that said Letters Patent are hereby corrected as shown below: H

Y E HPN N no- =0 I Column 9, Lines 26-62, 2 delete the formula and substitute therefore:

9 Y H-U HO- =0 0 HOX Signed and Scaled this Fourth D 3y of March 1980 [SEA L] A nest:

SIDNEY A. DIAMOND Arresting Oficer Commissioner of Patents and Trademarks 

1. A COMPOUND HAVING THE FORMULA
 2. A compound as in claim 1, poly-5-fluoro-2''-fluoro-2''-deoxyuridylic acid.
 3. A compound as in claim 1, poly-5-fluoro-2''-chloro-2''-deoxyuridylic acid.
 4. A compound as in claim 1, poly-5-fluoro-2''-bromo-2''-deoxyuridylic acid.
 5. A compound as in claim 1, poly-5-chloro-2''-fluoro-2''-deoxyuridylic acid.
 6. A compound as in claim 1, poly-5-chloro-2''-chloro-2''-deoxyuridylic acid.
 7. A compound as in claim 1, poly-5-chloro-2''-bromo-2''deoxyuridylic acid.
 8. A compound as in claim 1, poly-5-bromo-2''-fluoro-2''-deoxyuridylic acid.
 9. A compound as in claim 1, poly-5-bromo-2''-chloro-2''deoxyuridylic acid.
 10. A compound as in claim 1, poly-5-bromo-2''-bromo-2''-deoxyuridylic acid.
 11. A compound as in claim 1, poly-5-iodo-2''-fluoro-2''-deoxyuridylic acid.
 12. A compound as in claim 1, poly-5-iodo-2''-chloro-2''-deoxyuridylic acid.
 13. A compound as in claim 1, poly-5-iodo-2''-bromo-2''-deoxyuridylic acid. 